Trinucleotide Repeat Instability via DNA Damage and Repair

描述（由申请人提供）：遗传性三核苷酸重复序列（TNR）不稳定性（即扩增和缺失/收缩）与40多种人类家族性神经退行性疾病和癌症相关。非遗传性体细胞TNR不稳定性可能参与了这些疾病在普通公众中的发展。目前还没有有效的治疗TNR相关疾病的方法，部分原因是对TNR的潜在机制缺乏了解。我们最近发现DNA碱基损伤和碱基切除修复（BER）通过诱导单链DNA（ssDNA）断裂和促进GC自身碱基配对发夹的形成来启动和调节体细胞CAG重复扩增和缺失。这表明DNA碱基损伤、ssDNA断裂和BER在调节TNR不稳定性中的新作用。为了探索DNA损伤和BER作为预防和治疗TNR相关疾病的新靶点的潜力，在本项目中，我们试图了解环境和化疗诱导的ssDNA断裂及其低效修复如何参与BER期间的体细胞TNR不稳定性。这一目标将通过三个具体目标来实现。目的1是确定环境和化疗诱导的DNA碱基损伤和ssDNA断裂的积累是否可以以位点特异性方式优先导致CAG重复不稳定。将确定由环境毒物和化疗剂（如氯乙烯和替莫唑胺）诱导的CAG/CTG重复序列中ssDNA断裂的位点特异性累积。DNA损伤剂诱导的ssDNA断裂累积的独特模式将与重复扩增和缺失相关，以确定对CAG重复不稳定性的损伤特异性“位置效应”。在BER酶和辅因子的不平衡水平下，将进一步检查这些影响，以确定TNR不稳定性是否可以通过损害BER效率来调节。目的2是检验无效BER通过促进多个非B型DNA结构的形成而促进CAG重复缺失的假设。这将通过确定DNA聚合酶（Pol κ遗传变体，Pol κ）的低效DNA合成是否可以促进模板发夹的积累并促进TNR缺失来完成。目的3是确定是否可以通过BER蛋白质-蛋白质相互作用和功能协调有效地破坏非B型DNA结构来防止TNR扩增和缺失。本项目通过剖析环境和化疗DNA损伤、BER和TNR不稳定性之间的相互作用，探讨DNA损伤诱导的体细胞TNR不稳定性的基本机制。这些结果将为暴露于环境和化疗压力如何影响普通人群中TNR相关人类疾病的发展和进展以及如何通过DNA损伤修复预防这些不良影响提供重要的新见解。这将有助于确定新的TNR相关疾病的预防，诊断和治疗的目标，并为环境和化疗诱导的遗传毒性效应的风险评估提供新的信息。

DESCRIPTION (provided by applicant): Inherited trinucleotide repeat (TNR) instability, (i.e. expansions and deletions/contractions) is associated with more than 40 human familial neurodegenerative diseases and cancer. Non-inherited somatic TNR instability may be involved in the development of these diseases in the general public. No effective treatment for TNR- related diseases is yet available, partially because of a poor understanding of the underlying mechanisms. We have recently discovered that DNA base damage and base excision repair (BER) initiate and modulate somatic CAG repeat expansion and deletion by inducing single-strand DNA (ssDNA) breaks and promoting the formation of GC self-base-pairing hairpins. This indicates a new role of DNA base lesions, ssDNA breaks and BER in modulating TNR instability. To explore the potential of DNA damage and BER as new targets for the prevention and treatment of TNR-related diseases, in this project we seek to understand how environmentally and chemotherapeutically induced ssDNA breaks and their inefficient repair are involved in somatic TNR instability during BER. This goal will be achieved by pursuing three Specific Aims. Aim 1 is to determine if the accumulation of environmentally and chemotherapeutically induced DNA base lesions and ssDNA breaks can preferentially lead to CAG repeat instability in a site-specific manner. Site-specific accumulation of the ssDNA breaks in CAG/CTG repeat tracts induced by environmental toxicants and chemotherapeutic agents such as vinyl chloride and temozolomide will be determined. The unique patterns of ssDNA break accumulation induced by DNA-damaging agents will be correlated with repeat expansion and deletion to identify damage- specific "position effects" on CAG repeat instability. The effects will be further examined under imbalanced levels of BER enzymes and cofactors to determine if TNR instability can be modulated by compromised BER efficiency. Aim 2 is to test the hypothesis that inefficient BER facilitates CAG repeat deletion by promoting the formation of multiple non-B-form DNA structures. This will be done by determining if inefficient DNA synthesis by DNA polymerases (Pol ß genetic variants, Pol κ) can facilitate the accumulation of a template hairpin and promote TNR deletion. Aim 3 is to determine if TNR expansion and deletion can be prevented by efficiently disrupting non-B-form DNA structures through BER protein-protein interactions and functional coordination. This project addresses the fundamental mechanisms underlying DNA damage-induced somatic TNR instability by dissecting the interplay among environmental and chemotherapeutic DNA damage, BER, and TNR instability. The results will provide important new insights into how exposure to environmental and chemotherapeutic stresses may influence the development and progression of TNR-related human diseases in the general population, and how these adverse effects can be prevented by DNA damage repair. This will help to identify novel targets for prevention, diagnosis, and treatment of TNR-related diseases, and provide new information for risk assessment of environmentally and chemotherapeutically induced genotoxic effects.